Mammalian cancer cell and transgenic mammal carrying human protooncogene and kit for diagnosing cancer using said protooncogene

ABSTRACT

The present invention provides a mammalian cell transfected with an expression vector comprising a human protooncogene which has the nucleotide sequence of SEQ ID NO: 1; a mammalian embryo carrying a nucleic acid construct comprising the human protooncogene; a transgenic cancered mammal, which is derived from the mammalian embryo; and a kit for diagnosing a cancer selected from the group consisting of breast, kidney, ovary and stomach cancers, which comprises a probe having the nucleotide sequence complementary to mRNA transcribed from the human protooncogene or a portion of the mRNA; or an antibody binding specifically to a protein translated from the mRNA or a portion of the protein.

FIELD OF THE INVENTION

[0001] The present invention relates to a mammalian cancer celltransformed with an expression vector comprising a human protooncogenehaving the nucleotide sequence of SEQ ID NO: 1; a mammalian embryocomprising a nucleic acid construct containing the protooncogene; atransgenic cancered mammal derived from the mammalian embryo; and a kitfor diagnosing breast, kidney, ovary or stomach cancer using theprotooncogene.

BACKGROUND OF THE INVENTION

[0002] Higher animals including man each carry approximately 100,000genes, but only about 15% thereof is expressed, and characteristics ofindividual's biological process, e.g., genesis, differentiation,homeostasis, responses to stimuli, control of cell cycle, aging andapoptosis (programmed cell death), are determined depending on whichgenes are expressed (Liang, P. and A. B. Pardee, Science, 257: 967-971(1992)).

[0003] Pathogenic phenomena such as tumorigenesis are caused by genemutation which brings about changes in the mode of gene expression.

[0004] It has been reported that tumorigenesis is caused by variousgenetic changes such as the loss of chromosomal heterozygosity,activation of oncogenes and inactivation of tumor suppressor genes,e.g., p53 gene (Bishop, J. M., Cell, 64, 235-248 (1991); and Hunter, T.,Cell, 64, 249-270 (1991)). Further, it has been reported that 10 to 30%of human cancer arises from the activation of oncogene throughamplification of protooncogenes.

[0005] Therefore, the activation of protooncogenes plays an importantrole in the etiology of many tumors and there has existed a need toidentify protooncogenes.

[0006] The present inventor reported that a protooncogene, humancervical cancer 1 (HCCR-1), is specifically overexpressed in cancercells (Korean Patent Laid-open Publication No. 2001-39566). In order tounravel the mechanism involved in the tumorigenesis, the presentinventor has endeavored to prepare a cancer cell line and a transgenicmammal developing cancer using HCCR-1 protooncogene.

SUMMARY OF THE INVENTION

[0007] Accordingly, it is an object of the present invention to providea mammalian cell having introduced HCCR-1 protooncogene.

[0008] Another object of the present invention is to provide a mammalianembryo carrying HCCR-1 protooncogene, and a transgenic mammal derivedfrom the mammalian embryo.

[0009] A further object of the present invention is to provide a kit fordiagnosing breast, kidney, ovary or stomach cancer using HCCR-1protooncogene.

[0010] In accordance with one aspect of the present invention, there isprovided a mammalian cell transformed with an expression vectorcomprising human protooncogene HCCR-1 which has the nucleotide sequenceof SEQ ID NO: 1.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The above objects and features of the present invention willbecome apparent from the following description of preferred embodimentstaken in conjunction with the accompanying drawings, in which:

[0012]FIGS. 1A and 1B are the respective phase-contrast features ofmonolayer-cultured HCCR-1M and parental wild-type NIH/3T3 cells;

[0013]FIG. 2 is the hematoxylin-eosin staining result ofmonolayer-cultured HCCR-1M cells;

[0014]FIG. 3 is a transmission electron microscope picture of HCCR-1Mcells;

[0015]FIG. 4 is a photograph of the nude mouse allografted with HCCR-1Mcells showing the palpable tumor nodule;

[0016]FIG. 5 is the hematoxylin-eosin staining result of the palpabletumor nodules taken from the nude mouse allografted with HCCR-1M cells;

[0017]FIG. 6 is a transmission electron microscope picture of thepalpable tumor nodules taken from the nude mouse allografted withHCCR-1M cells;

[0018]FIGS. 7A to 7D are the immunohistochemical analysis resultsshowing the expression of reticulin fibers, keratin, epithelial membraneantigen and vimentin, respectively, in the palpable tumor nodules takenfrom the nude mouse allografted with HCCR-1M cells;

[0019]FIG. 8 is a phase-contrast feature of monolayer-cultured HCCR-1MNcells;

[0020]FIG. 9 is the western blot analysis result showing the expressionof HCCR-1 protooncogene in HCCR-1M and HCCR-1MN cells;

[0021]FIGS. 10A and 10B are the phase-contrast features ofmonolayer-cultured HCCR-1H and parental wild-type 293 cells,respectively;

[0022]FIG. 11 is the hematoxylin-eosin staining result of the palpabletumor nodules taken from the nude mouse xenografted with HCCR-1H cells;

[0023]FIGS. 12A to 12C are the immunohistochemical analysis resultsshowing the expression of cytokeratin 8, cytokeratin 19 and vimentin,respectively, in the palpable tumor nodules taken from the nude mousexenografted with HCCR-1H cells;

[0024]FIG. 13 is the northern blot analysis result showing theexpression of HCCR-1 protooncogene in HCCR-1H cells;

[0025]FIG. 14 is a graph showing protein kinase C activities of parentalwild-type NIH/3T3 cell, comparative NIH/3T3 cell transfected with vectorpcDNA3 and HCCR-1M cell;

[0026]FIG. 15 is a graph showing telomerase activities of positivecontrol 293 cell, negative control cell, parental wild-type NIH/3T3cell, comparative NIH/3T3 cell transfected with vector pcDNA3 andHCCR-1M cells;

[0027]FIGS. 16A to 16C are the northern blot analysis results showingthe expression of egr-1, c-fos and glyceraldehyde-2-phosphatedehydrogenase (GAPDH) genes, respectively, in HCCR-1M cell;

[0028]FIG. 17A is the western blot analysis result showing theexpression of p53 tumor suppressor gene in HCCR-1H cell; and FIG. 17B,β-actin of the same blot;

[0029]FIG. 18 is the immunoprecipitation result showing the expressionof p53 tumor suppressor in HCCR-1H cell;

[0030]FIGS. 19A, 19C, 19E, 19G and 19I are the western blot analysisresults showing the expression of MDM2, Bax, p21WAF1, p16INK4A andp14^(ARF) genes, respectively, in HCCR-1H cell; and FIGS. 19B, 19D, 19F,19H and 19J, β-actin in each of the above blots;

[0031]FIG. 20A is the western blot analysis result showing GST proteinin the precipitate prepared by immunoprecipitating the FLAG protein ofthe CHO cell cotransfected with the vector expressing the fusion proteinof FLAG and HCCR-1 and the vector expressing the fusion protein of GSTand D52; and FIG. 20B, PLAG protein in the precipitate prepared byimmunoprecipitating the GST protein;

[0032]FIG. 21 is a schematic diagram of the nucleic acid constructcontaining HCCR-1 protooncogene;

[0033]FIG. 22 is a transgenic cancered mouse derived from an embryohaving introduced HCCR-1 protooncogene;

[0034]FIG. 23 is a photograph of breast tumor of the transgenic mousederived from the embryo carrying introduced HCCR-1 protooncogene;

[0035]FIG. 24 is the hematoxylin-eosin staining result of the breasttumor taken from a transgenic mouse derived from the embryo carryingintroduced HCCR-1 protooncogene;

[0036]FIG. 25 is a transmission electron microscope picture of thebreast tumor taken from the transgenic mouse derived from the embryocarrying introduced HCCR-1 protooncogene;

[0037]FIG. 26A is the northern blot analysis result showing theexpression of HCCR-1 protooncogene in breast, kidney, ovary and stomachtumor tissues; and FIG. 26B, the same blot hybridized with β-actinprobe;

[0038]FIG. 27 is the western blot analysis result showing the expressionof HCCR-1 protooncogene in human breast, kidney, ovary and stomach tumortissues; and

[0039]FIG. 28 is the fluorescence in situ hybridization analysis resultshowing the position of HCCR-1 protooncogene on the human chromosome.

DETAILED DESCRIPTION OF THE INVENTION

[0040] The human protooncogene (hereinafter “HCCR-1 protooncogene”) ofSEQ ID NO: 1 which is used in the preparation of the inventive mammaliancell and transgenic mammal is located on the long arm (12q) of the 12thchromosome and has an open reading frame which encodes a protein havingthe amino acid sequence of SEQ ID NO: 2 and molecular weight of about 50kDa.

[0041] In consideration of the degeneracies of codons and the preferredcodons in a specific animal wherein the HCCR-1 protooncogene is to beexpressed, various changes and modifications of the nucleotide sequenceof SEQ ID NO: 1 may be made, e.g., in the coding region thereof withoutadversely altering the amino acid sequence of the expressed protein, orin the non-coding region without adversely affecting the expression ofthe HCCR-1 protooncogene. Therefore, the present invention alsoincludes, in its scope, a polynucleotide having substantially the samebase sequence as the HCCR-1 protooncogene, and a fragment thereof. Asused herein, “substantially the same polynucleotide” refers to apolynucleotide whose base sequence shows 80% or more, preferably 90% ormore, most preferably 95% or more homology to the HCCR-1 protooncogene.

[0042] The HCCR-1 protooncogene can be obtained from human cancertissues or synthesized using a conventional DNA synthesis method.Further, the HCCR-1 protooncogene thus prepared may be inserted to aconventional vector to obtain an expression vector.

[0043] The expression vector may, in turn, be introduced into a suitablemammalian cell to obtain a mammalian cell transfected with theexpression vector. For example, NIH/3T3 cells (ATCC CRL 1658), adifferentiated mouse fibroblast cell line, and 293 cells (ATCC CRL1573), a human embryonic kidney cell line, were transfected withexpression vector pcDNA3/HCCR-1 containing the HCCR-1 protooncogene toobtain the NIH/3T3 and 293 cells transfected with HCCR-1 protooncogene,designated HCCR-1M and HCCR-1H cells, respectively, which were depositedwith Korean Collection for Type Cultures (KCTC) (Address: #52, Oun-dong,Yusong-ku, Taejon 305-333, Republic of Korea) on Dec. 26, 2000 under theaccession numbers of KCTC 0923BP and KCTC 0922BP, respectively, inaccordance with the terms of Budapest Treaty on the InternationalRecognition of the Deposit of Microorganism for the Purpose of PatentProcedure.

[0044] The inventive mammalian cell thus obtained overexpresses HCCR-1protooncogene and exhibits morphological characteristics of tumor giantcells. Specially, mouse cell HCCR-1M has a polygonal shape, lobulatednucleus with prominent nucleoli, well developed rough endoplasmicreticula (rER) and Golgi complexes, and microvilli on the cell surface,and exhibits atypical mitotic figures. The human cell HCCR-1H hasincreased cell dimension and cellularity compared with the wild-typecell.

[0045] The inventive mammalian cell has a tumorigenicity and therefore amammal grafted with the inventive mammalian cell develops a tumor, e.g.,palpable tumor. Further, a mammalian cell isolated from the tumor has atumorigenicity. For example, a mouse cell, designated HCCR-1MN, isolatedfrom a palpable tumor of a nude mouse grafted with HCCR-1M cell has themorphological features similar with those of HCCR-1M cell.

[0046] The inventive mammalian cell produces a large amount of HCCR-1protein which interacts with D52 protein (GenBank accession numberNM003288) which has been reported to be associated with breast carcinoma(Byrne, J. A., et al., Cancer Res., 55, 2896-2903 (1995); and Byrne, J.A., et al., Oncogene, 16, 873-881 (1998)). It also enhances a cellularprotein kinase C (PKC) and telomerase activities, induces the loss ofparticular cell cycle checkpoint controls, and suppresses the egr-1expression, thereby predisposing cells to malignant conversion. In thisprocess, the expression of p53 tumor suppressor gene involved in thecell cycle control increases while most of the p53 proteins areinactivated, thereby leading to malignant conversion. The HCCR-1protooncogene induces functional inactivation of p53 tumor suppressor atan upstream level. Therefore, if a portion of HCCR-1 protooncogene whichregulates p53 gene is identified, the p53 activity may be recovered bychanging the corresponding nucleotide sequence.

[0047] Further, a nucleic acid construct comprising HCCR-1 protooncogenemay be introduced into a suitable embryo of a mammal, e.g., a mouse, andthe mammalian embryo thus obtained may be implanted in the uterus of asuitable recipient and allowed to develop to term to obtain a transgenicmammal. The method for preparing a transgenic mammal is well known inthe art, and it is preferable to introduce the nucleic acid constructinto an embryo at the 8-cell stage or earlier.

[0048] The transgenic mammal thus prepared overexpresses HCCR-1protooncogene and develops ductal papillary adenocarcinoma of breastexhibiting the charachteristic features of: well-circumscribed nodulewithout capsule; papillary structures and glandular or duck-likestructures; extensive necrosis occurring in the central portion; cuboidor ovoid cells having indistinct borders; normal breast tissues beingadjacent thereto; and a significant amount of granular, eosinophiliccytoplasm.

[0049] A transgenic embryo may be obtained by mating male and femaletransgenic mammals. An exemplary transgenic embryo is mouse FVB/N embryo(designated mouse embryo HCCR-1) carrying the nucleic acid constructhaving the structure of FIG. 21, which was deposited with the KoreanCollection for Type Cultures (KCTC) (Address: #52, Oun-dong, Yusong-ku,Taejon 305-333, Republic of Korea) on Dec. 26, 2000 under the accessionnumber of KCTC 0924BP, in accordance with the terms of Budapest Treatyon the International Recognition of the Deposit of Microorganism for thePurpose of Patent Procedure.

[0050] Since the inventive mammalian cell and transgenic mammaloverexpresses HCCR-1 protooncogene to develop cancer, they areadvantageously used in screening a carcinogen or anti-cancer agent,e.g., antioxidant.

[0051] Further, HCCR-1 protooncogene is overexpressed in breast, kidney,ovary and stomach cancers, and therefore, the product thereof may beeffectively used in diagnosing the cancers. The cancer diagnosis may beconducted by reacting mRNA or a protein sample taken from breast,kidney, ovary or stomach tissue of a subject with a probe or antibodywhich specifically binds to mRNA or the protein expressed from theHCCR-1 protooncogene; and detecting the HCCR-1 mRNA or protein accordingto various methods known in the art, thereby determining whether thesubject has overexpressed products of HCCR-1 protooncogene. The presenceof the protooncogene product may be easily detected by labeling theprobe or antibody with a radioisotope or an enzyme. Therefore, thepresent invention also provides a kit for diagnosing a breast, kidney,ovary or stomach cancer, which comprises a probe or an antibody whichspecifically binds to an mRNA or protein expressed from HCCR-1protooncogene. Exemplary probes include a polynucleotide oroligonucleotide which has a nucleotide sequence complementary to mRNAtranscribed from the HCCR-1 protooncogene or a portion of the mRNA sothat it specifically binds to the HCCR-1 mRNA, and preferred is thathaving the nucleotide sequence of SEQ ID NO: 3. The probe can beobtained from human tissues or synthesized using a conventional DNAsynthesis method. Further, the antibody can be prepared according toconventional method kwon in the art.

[0052] The following Examples are intended to further illustrate thepresent invention without limiting its scope.

REFERENCE EXAMPLE 1 Transmission Electron Microscopy (TEM)

[0053] Cells or tissues were fixed with 2.5% glutaraldehyde in aphosphate buffer (pH 7.4) and then postfixed with a 2% osmium tetroxide.Specimens were dehydrated in a graded series of ethanols and embedded inEpon 812. Ultrathin sections thereof were stained with uranyl acetateand lead citrate, and photographed by TEM (JEOL 1,200 EX, Tokyo, Japan).

REFERENCE EXAMPLE 2 Western Blot Analysis

[0054] Cells were harvested and lysed in a Laemmli sample buffer inaccordance with the method described by Laemmli (Nature, 227, 680-685(1970)). The cellular proteins were separated by 10% SDS-PAGE and thenelectroblotted onto nitrocellulose membranes. The membranes wereincubated with the antibody. After washing, the membranes were incubatedwith a blocking solution containing 1:1,000 dilution ofperoxidase-conjugated goat anti-rat immunoglobulin (JacksonImmunoResearch) as a secondary antibody. Proteins were revealed by anECL-Western blot detection kit (Amersham).

REFERENCE EXAMPLE 3 Immunohistochemical Analysis

[0055] Tissues were incubated with a primary antibody and then cut in athickness of 5 μm. Binding of primary antibody was visualized bybiotinylated secondary antibody, avidin, biotinylated horseradishperoxidase and AEC (aminoethyl carbazole) as the chromogen(HISTOSTAIN-BULK KITS, Zymed).

REFERENCE EXAMPLE 4 Isolation of Total RNA

[0056] Total RNAs were extracted from the tissue specimens or cellsusing a commercial system (RNeasy total RNA kit, Qiagen Inc., Germany),and DNA contaminants were removed therefrom using Message clean kit(GenHunter Corp., USA).

REFERENCE EXAMPLE 5 Northern Blot Analysis

[0057] 20 μg of the total RNAs were electrophoresed through 1%formaldehyde agarose gel and transferred to nylon membranes(Boehringer-Mannheim, Germany). The blot was hybridized overnight at 42°C. with ³²P-labeled random-primed probe which was prepared using arediprime II random prime labeling system (Amersham, UK). The northernblot analysis results were consistently repeated two times, asquantified by densitometry and the same blot was hybridized with aβ-actin probe to confirm mRNA integrity.

PREPARATION EXAMPLE 1 Construction of Expression Vector ContainingHCCR-1 Protooncogene

[0058]E. coli JM-109/HCCR1 (KCTC 0667BP) has expression vectorpCEV-LAC/HCCR-1 containing the full length HCCR-1 cDNA represented bythe nucleotide sequence of SEQ ID NO: 1. E. coli JM-109/HCCR1 cells werecultured and expression vector pCEV-LAC/HCCR-1 was isolated therefrom.Expression vector pCEV-LAC/HCCR-1 thus obtained was cleaved with SalI toobtain a 2.1 kb DNA fragment containing the full length HCCR-1 cDNA.

[0059] Mammal expression vector pcDNA3 (Invitrogene) was cleaved withXhoI to make a compatible end with SalI. The 2.1 kb SalI fragment wasligated with the XhoI-digested pcDNA3 to obtain expression vectorpcDNA3/HCCR-1 containing the full length HCCR-1 cDNA.

PREPARATION EXAMPLE 2 Production of Anti-HCCR-1 Antibody

[0060] In order to obtain HCCR-1 proteins useful in the preparation ofanti-HCCR-1 antibody, E. coli JM-109/HCCR1 (KCTC 0667BP) cells werecultured and expression vector pCEV-LAC/HCCR-1 was isolated therefrom.Expression vector pCEV-LAC/HCCR-1 was subjected to polymerase chainreaction (PCR) using primers of SEQ ID Nos: 4 and 5 to amplify a DNAregion from the 123rd to the 473rd nucleotides of SEQ ID NO: 1 (the 39thto the 155th amino acid residues of SEQ ID NO: 2). The PCR product thusobtained was inserted at BamHI/SalI sites of vector pMAL-p2 (New EnglandBiolab, USA) to obtain a recombinant vector (designatedpMAL-p2/HCR-1-c-terminal), and then, E. coli BL21 (ATCC CRL 47092) wastransformed with recombinant vector pMAL-p2/HCCR-1-c-terminal. Thetransformant was cultured in LB media and the resulting culture wasdiluted with 100-fold volume of LB media. The diluted culture wasincubated for 3 hours, and then, 1 mM isopropylβ-D-thiogalacto-pyranoside (IPTG, Sigma) was added thereto to induceexpression of HCCR-1 protooncogene. The C-terminal portion of HCCR-1protein fused with maltose binding protein (MBP, 42 kDa) derived fromvector pMAL-p2 was expressed which has the molecular weight ofapproximate 64 kDa. The 64 kDa fusion protein was purified from theculture using pMAL protein fusion and purification system (New EnglandBiolab., USA).

[0061] Then, ten 3-month-old New Zealand white rabbits each weighingabout 2.5 kg were intraperitoneally administered with 1 mg of the fusionprotein weekly for 3 times. The blood sample was obtained from theimmunized rabbits and centrifuged to obtain a serum containing apolyclonal antibodies.

EXAMPLE 1 Preparation of Mouse Cell Transfected with HCCR-1Protooncogene

[0062] NIH/3T3 cells (ATCC CRL 1658), differentiated mouse fibroblastcell line, were transfected with expression vector pcDNA3/HCCR-1obtained in Preparation Example 1 using Lipofectamine (Gibco BRL), andthe resulting NIH/3T3 cells were cultured in Waymouth MB 752/1 mediumsupplemented with G418 (Gibco) to obtain the NIH/3T3 cell transfectedwith HCCR-1 protooncogene, designated HCCR-1M, which was deposited withKorean Collection for Type Cultures on Dec. 26, 2000 under the accessionnumber of KCTC 0923BP.

EXAMPLE 2 Preparation of Human Cell Transfected with HCCR-1Protooncogene

[0063] The procedure of Example 1 was repeated except that 293 cells(ATCC CRL-1573) were used instead of NIH/3T3 cells, to obtain the 293cell transfected with HCCR-1 protooncogene, designated HCCR-1H, whichwas deposited with Korean Collection for Type Cultures on Dec. 26, 2000under the accession number of KCTC 0922BP.

COMPARATIVE EXAMPLE Preparation of Comparative Cell Transfected withVector pcDNA3

[0064] The procedure of Example 1 was repeated except that vector pcDNA3(Invitrogen) was used instead of vector pcDNA3/HCCR-1 to obtaincomparative cells transfected with vector pcDNA3.

EXAMPLE 3 Morphological Characteristics of HCCR-1 M Cell

[0065] (Step 1) Phase-contrast features of HCCR-1M cell

[0066] HCCR-1M cells obtained in Example 1 were cultured in monolayer inWaymouth MB 752/1 medium and the resulting HCCR-1M cells were examinedunder the phase-contrast microscope. The parental wild-type NIH/3T3cells were used as a control.

[0067]FIGS. 1A and 1B are the respective phase-contrast features ofmonolayer-cultured HCCR-1M and parental wild-type NIH/3T3 cells. As canbe seen from FIGS. 1A and 1B, the parental wild-type NIH/3T3 cells arespindle shaped cells having a long slender nucleus and a scanty amountof cytoplasm, while HCCR-1M cells have a polygonal shape with an ovoidnucleus and plump cytoplasm.

[0068] (Step 2) Hematoxylin-eosin staining

[0069] HCCR-1M cells obtained in Example 1 were cultured in monolayer inWaymouth MB 752/1 medium and the resulting HCCR-1M cells were stainedwith hematoxylin-eosin.

[0070]FIG. 2 is the hematoxylin-eosin staining result ofmonolayer-cultured HCCR-1M cells. As can be seen from FIG. 2, HCCR-1Mcells exhibit nuclear pleiomorphism, distinct nucleoli, granularchromatin patterns, tumor giant cells and atypical mitotic figures.

[0071] (Step 3) TEM

[0072] HCCR-1M cells obtained in Example 1 were subjected to TEMaccording to the procedure of Reference Example 1.

[0073]FIG. 3 is the TEM picture of HCCR-1M cell wherein the scale barrepresents the length of 3 μm and the inset is higher magnification ofarea indicated by circle (scale bar, 1 μm). As can be seen from FIG. 3,the HCCR-1M cells have microvilli on the cell surface, lobulated nucleuswith prominent nucleoli and well-developed rough endoplasmic reticula(rER) and Golgi complexes (circle).

[0074] The above results show that HCCR-1M cell has morphologicalcharacteristics of tumor giant cell.

EXAMPLE 4 Tumorigenicity of HCCR-1M cell

[0075] 5×10⁶ each of HCCR-1M cells obtained in Example 1 were injectedsubcutaneously into the posterior lateral aspect of the trunk of 9 nudemice (5-week-old athymic nu/nu on BALB/c background).

[0076] All 9 nude mice injected with HCCR-1M cells showed palpabletumors after 21 days. FIG. 4 is the photograph of the nude mouseallografted with HCCR-1M cells. This suggests that the HCCR-1M cellshave a tumorigenicity.

[0077] When the subcutaneous tumors reached 1.5-2.5 cm in diameter, thenude mice were sacrificed and the tumors were removed therefrom whichwas used in the following tumor analysis and establishment of cell line.

EXAMPLE 5 Characteristics of Tumor Nodule of Nude Mouse Grafted withHCCR-1M Cell

[0078] Characteristics of nude mouse's tumor nodules induced by HCCR-1Mallograft were examined as follows.

[0079] (Step 1) Hematoxylin-eosin staining

[0080] The tumor nodules obtained in Example 4 were stained withhematoxylin-eosin staining.

[0081]FIG. 5 is the hematoxylin-eosin staining result (×250) of thetumor nodules. As can be seen from FIG. 5, the tumor nodules havetypical epithelial cell nests separated by fibrous stroma.

[0082] (Step 2) TEM

[0083] The tumor nodules obtained in Example 4 were subjected to TEMaccording to the procedure of Reference Example 2.

[0084]FIG. 6 is the TEM picture of the tumor nodules wherein the scalebar has the length of 3 μm and the inset is higher magnification of areaindicated by circle (scale bar, 0.5 μm). As can be seen from FIG. 6, thecells of tumor nodules have well-developed organelles and are tightlyconnected by desmosomes. This suggests that the HCCR-1M cells werechanged into epidermal cells.

[0085] (Step 3) Immunohistochemical analysis

[0086] The tumor nodules obtained in Example 4 were subjected toimmunohistochemistry according to the procedure of Reference Example 3using each of anti-reticulin fiber, anti-keratin, anti-EMA (epithelialmembrane antigen) and anti-vimentin antibodies (DAKO) as a primaryantibody.

[0087]FIGS. 7A to 7D are the immunohistochemical analysis results (×250)showing the expression of reticulin fibers, keratin, EMA and vimentin,respectively, in the tumor nodules. As can be seen from FIGS. 7A to 7D,the cell nests were enveloped by reticulin fibers (FIG. 7A) and thecells showed coexpression of epithelial markers, such as keratin (FIG.7B) and EMA (FIG. 7C) and of the mesenchymal marker, vimentin (FIG. 7D).These results suggest that HCCR-1 protooncogene caused the conversion ofthe mesenchymal NIH/3T3 cells to epithelial cells.

[0088] Particularly, the mesenchymal NIH/3T3 cell introduced with anoncogene transforms into sarcoma, whereas the nude mouse allograftedwith HCCR-1M cell develops a palpable tumor.

EXAMPLE 6 Preparation of Cancer Cell Line from Nude Mouse Allograftedwith HCCR-1M cell

[0089] Cells were isolated from the tumor nodules obtained in Example 4and then cultured in Waymouth MB 752/1 medium supplemented with 20%bovine fetal serum, which were designated HCCR-1MN cell.

EXAMPLE 7 Characteristics of HCCR-1MN Cells

[0090] The HCCR-1MN cells obtained in Example 6 were subjected tophase-contrast microscopy by repeating the procedure of Step 1 ofExample 3.

[0091]FIG. 8 is the phase-contrast feature (×300) of monolayer-culturedHCCR-1MN cells. As can be seen from FIG. 8, HCCR-1MN cells havemorphological characteristics similar with those of HCCR-1M cellsrepresented in Step 1 of Example 3.

EXAMPLE 8 Western Blot Analyses of HCCR-1M and HCCR-1MN Cells

[0092] To examine expression of HCCR-1 protooncogene in HCCR-1M andHCCR-1MN cells, HCCR-1M and HCCR-1MN cells obtained in Examples 1 and 6,respectively, were subjected to western blot analysis according to theprocedure of Reference Example 2 using the anti-HCCR-1 serum obtained inPreparation Example 2. For the comparison, the parental wild-typeNIH/3T3 cells and the comparative NIH/3T3 cells obtained in ComparativeExample were used in the western blot analysis.

[0093]FIG. 9 is the western blot analysis result showing the expressionof HCCR-1 protooncogene in HCCR-1M and HCCR-1MN cells. As can be seenfrom FIG. 9, the HCCR-1 protein is overexpressed in HCCR-1M and HCCR-1MNcells, but not in the parental wild-type NIH/3T3 cells and comparativeNIH/3T3 cells transfected with vector pcDNA3, as represented as weakband.

EXAMPLE 9 Characteristics of HCCR-1H Cell

[0094] To examine morphological characteristics of HCCR-1H cell, theHCCR-1H cells were subjected to phase-contrast microscopy according tothe procedure of Step 1 of Example 3. The parental wild-type 293 cellswere used as a control.

[0095]FIGS. 10A and 10B are the respective phase-contrast features(×300) of monolayer-cultured HCCR-1H and parental wild-type 293 cells.As can be seen from FIGS. 10A and 10B, the HCCR-1H cell has increasedcell dimension and cellularity compared with the wild-type 293 cell.

EXAMPLE 10 Tumorigenicity of HCCR-1H Cell

[0096] To examine tumorigenicity of HCCR-1H cell, the procedure ofExample 4 was repeated using HCCR-1H cells obtained in Example 2.

[0097] Nude mice xenografted with HCCR-1H cells showed palpable tumorsafter 21 days. This suggests that the HCCR-1H cell has a tumorigenicity.

[0098] When the tumors reached 1-1.5 cm in diameter, the nude mice weresacrificed and the tumors were removed therefrom which was used in thefollowing tumor analysis.

EXAMPLE 11 Characteristics of Tumor Nodule of Nude Mouse Grafted withHCCR-1H Cell

[0099] Characteristics of nude mouse's tumor nodules induced by HCCR-1Hallograft were examined as follows.

[0100] (Step 1) Hematoxylin-eosin staining

[0101] The tumor nodules obtained in Example 10 were stained withhematoxylin-eosin staining.

[0102]FIG. 11 is the hematoxylin-eosin staining result (×250) of thepalpable tumor nodules. As can be seen from FIG. 11, the tumor nodulehas characteristics of typical epithelial cell carcinomas, i.e., atypismof cell shape, pleomorphism, nucleolar enlargement, and increased volumeratio of nucleus to cytoplasm.

[0103] (Step 2) immunohistochemical analysis

[0104] The tumor nodules obtained in Example 10 were subjected toimmunohistochemistry according to the procedure of Reference Example 3using each of anti-cytokeratin-8, anti-cytokeratin-19 and anti-vimentinantibodies (DAKO) as a primary antibody.

[0105]FIGS. 12A to 12C are the immunohistochemical analysis results(×250) showing the expression of cytokeratin 8, cytokeratin 19 andvimentin, respectively, in the palpable tumor nodules. As can be seenfrom FIGS. 12A to 12C, the tumor cells showed coexpression of epithelialmarkers, such as cytokeratin-8 and -19 (FIGS. 12A and 12B), and themesenchymal marker, vimentin (FIG. 12C). These results suggest thatHCCR-1 protooncogene caused the conversion of the mesenchymal 293 cellsto epithelial cells.

EXAMPLE 12 Northern Blot Analysis of HCCR-1H Cell

[0106] To examine expression level of HCCR-1 protooncogene in HCCR-1Hcells, total RNAs were isolated from HCCR-1H cells obtained in Example 2according to the procedure of Reference Example 4 and subjected tonorthern blot according to the procedure of Reference Example 5 using³²P-labeled random HCCR-1 cDNA probe.

[0107]FIG. 13 is the northern blot result showing the expression ofHCCR-1 protooncogene in HCCR-1H and parental wild-type 293 cells. As canbe seen from FIG. 13, about 2.1 kb mRNA single transcript wasoverexpressed in HCCR-1H cells while not in the parental wild-type 293cells.

EXAMPLE 13 Tumorigenesis Mechanism Induced by HCCR-1 Protooncogene inMouse Cells

[0108] To examine the tumorigenesis mechanism induced by HCCR-1protooncogene in mouse cells, abnormalities of protein kinase C (PKC)and telomerase activities, loss of cell cycle checkpoint control,alteration of egr-1, c-fos and GAPDH expression, which have beenreported to be relevant to the tumorigenesis process, were assessed asfollows.

[0109] (Step 1) PKC activity assay

[0110] Generally, tumors develop secondarily to abnormalities inPKC-mediated signal transduction process. To ensure that HCCR-1modulates the protein kinase C (PKC) activity, PKC assay was performedusing parental wild-type NIH3T3 cells, comparative NIH/3T3 cellstransfected with vector pcDNA3 obtained in Comparative Example andHCCR-1M cells obtained in Example 1.

[0111] PKC activity was measured using the SigmaTECT™ Protein Kinase CAssay System (Promega) according to the manufacturer's instructions. PKCactivity was defined as the difference of the amounts of PKCincorporated into substrate per minute in the absence and presence ofphospholipids. Each value is the means ±s.d. of three independentexperiments.

[0112]FIG. 14 is the graph showing PKC activities of parental wild-typeNIH/3T3 cell, comparative NIH/3T3 cell transfected with vector pcDNA3and HCCR-1M cell. As can be seen from FIG. 14, the PKC activity ofHCCR-1M cells is about 10-fold higher than the wild-type. This suggeststhat HCCR-1 protooncogene enhances the cellular PKC activity.

[0113] (Step 2) Telomerase activity assay

[0114] Based on the report that PKC induces a marked increase intelomerase activity, telomerase activity assay was performed usingwild-type NIH/3T3 cells, comparative NIH/3T3 cells transfected withvector pcDNA3 obtained in Comparative Example and HCCR-1M cells obtainedin Example 1.

[0115] Telomerase activity was measured using the telomerase PCR-ELISAkit (Boehringer Mannheim, USA) according to the manufacturer'sinstructions. Human telomerase-positive immortalized human kidney cellsprovided in the kit were used as a positive control. Used as a negativecontrol was the 293 cells pretreated with RNase. Assays were performedwith an extract amount equivalent to 1×10³ cells. Results show theaverage mean optical density (OD) values from four separate experiments(means ±s.d.).

[0116]FIG. 15 is the graph showing telomerase activities of positivecontrol cell, negative control cell, parental wild type NIH/3T3 cell,comparative NIH/3T3 cell transfected with vector pcDNA3 and HCCR-1Mcells. As can be seen from FIG. 15, the wild-type NIH/3T3 cells showeddetectable telomerase activity while HCCR-1M cells have the increasedtelomerase activity by a factor of about 7 as compared with the parentalwild-type cells. The telomerase activity of the positive control cell ishigh while that of the negative control cells is not detectable. Theseresults are consistent with the previous study (Holt, S. E., Wright, W.E. and Shay J. W. Mol. Cell Biol. 16, 2932-2939 (1996)), suggesting thatHCCR-1M cell has high telomerase activity induced by activated PKC.

[0117] (Step 3) Cell cycle test

[0118] Tumor cells typically have acquired damage to genes that directlyregulate their cell cycles. In order to examine whether there was analternation in growth properties of HCCR-1M cells, the cell cycleprofiles was determined as follows.

[0119] HCCR-1M and parental wild-type NIH/3T3 cells cultured at mid-logphase were growth arrested by incubation in a DMEM medium containing0.5% fetal calf serum for 36 hours. Cells were treated with trypsin,harvested and fixed in 70% ethanol. 50 μg/ml of a propidium iodidestaining solution (Sigma) and 100 units per ml of RNase A (BoerhingerMannheim) were added to 2×10⁶ cells. After incubation for 30 min., thecellular DNA content was determined by fluorescence analysis at 488 nmusing a FACS Caliber (Becton Dickinson). A minimum of 1×10⁴ cells persample was analyzed with Modfit 5.2 software.

[0120] Percentage of parental wild-type NIH/3T3 and HCCR-1 cells inG₀/G₁, S and G₂/M phases were determined and the results are shown inTable I. TABLE I Wild-type NIH/3T3 HCCR-1M G₀/C₁ S G₂/M G₀/G₁ S G₂/MCell Percentage 55.7 20.6 24 46.6 31.5 22.4

[0121] As can be seen from Table I, the percentage of wild-type NIH/3T3and HCCR-1M cells in S-phase was 20.6% and 31.5%, respectively. Theseresults suggest that there was a significant shift of the cellpopulation out of the G₀/G₁-phase into the S-phase in HCCR-1M cells.

[0122] To assess the serum-dependent cell cycle progression, HCCR-1M andparental wild-type NIH/3T3 cells were cultured in DMEM medium containing0.5% fetal calf serum for 36 hours to arrest the growth. The cells weretransferred into DMEM medium containing 20% fetal calf serum andharvested at indicated times to determine percentage of cells in G₀/G₁,S and G₂/M phases. The results are shown in Table II. TABLE II CellPercentage Wild-type NIH/3T3 HCCR-1M Time (hour) G₀/C₁ S G₂/M G₀/G₁ SG₂/M 0 77 8.0 14.9 70 21.8 8.7 12 72.2 14.0 14.2 66.9 24.0 9.6 24 49.613.4 37.2 56.7 24.7 19.2 48 58.3 18.3 23.7 52.7 30.4 17.5

[0123] As can be seen from Table II, the percentage of parentalwild-type NIH/3T3 cells in S-phase at 0 hour was 8% while the percentageof HCCR-1M cells in S-phase was 21.8%. These results suggest thatconstitutive overexpression of HCCR-1 protooncogene allowed for arelative amount of resistance to serum deprivation-induced G₀/G₁ arrest.After transferring cells into DMEM medium containing 20% serum torelease cells from the growth arrest, there were consistent increases ofover 10% in the S-phase populations of HCCR-1M cells as compared towild-type NIH/3T3 cells. Therefore, overexpression of HCCR-1protooncogene could deregulate cell growth by shortening the G₀/G₁-phaseand increasing the S-phase population of cells.

[0124] (Step 4) Expression patterns of egr-1, c-fos and GAPDH

[0125] Based on the fact that expression patterns of egr-1, c-fos andGAPDH involve in tumor formation, expression patterns of egr-1, c-fosand GAPDH in HCCR-1M cells obtained in Example 1 were examined asfollows.

[0126] Growth of HCCR-1M and parental wild-type NIH/3T3 cells in mid-logphase were arrested by incubation in a DMEM medium containing 0.5% fetalcalf serum for 36 hours (quiescent period). The cells were transferredinto fresh DMEM medium containing 20% fetal calf serum and cultured for0, 15, 30, 60 and 120 min. to stimulate the growth (mitotic period).Total RNAs were isolated from quiescent and mitotic cells according tothe procedure of Reference Example 4 and subjected to northern blotaccording to the procedure of Reference Example 5 using ³²P-labeledrandom-primed egr-1, c-fos and GAPDH cDNA probe.

[0127]FIGS. 16A, 16B and 16C are the northern blot results showing theexpression of egr-1, c-fos and GAPDH, respectively, in HCCR-1H andparental wild-type NIH/3T3 cells. As can be seen from FIGS. 16A to 16C,mitotic HCCR-1M cells show a marked down-regulation of egr-1 expression,up-regulation of GAPDH and no significant difference of c-fos expressioncompared with the wild-type cells.

[0128] These results suggest that deregulation of HCCR-1 gene in mouseNIH/3T3 cells may result in the activation of PKC or telomerase, loss ofparticular cell cycle checkpoint controls, and downregulation of egr-1expression, thereby predisposing cells to malignant conversion.

EXAMPLE 14 Tumorigenesis Mechanism Induced by HCCR-1 Protooncogene inHuman Cell

[0129] To examine the tumorigenesis mechanism induced by HCCR-1protooncogene in a human cell, expression patterns of p53 tumorsuppressor, MDM2, Bax, p21WAF1, p16INK4A and p14^(ARF) genes, which havebeen reported to be relevant to the tumor, were tested as follows.

[0130] (Step 1) Expression pattern of p53 tumor suppressor

[0131] In order to examine the expression pattern of p53 in HCCR-1Hcell, western and northern blot analyses were conducted as follows.

[0132] HCCR-1H cells obtained in Example 2 and parental wild-type 293cells were lysed and the lysates were subjected to western blot usingmonoclonal anti-p53 antibody DO-7 (amino acids 37 to 45; NovocastraLaboratories, Ltd., UK) which reacts with wild-type p53 alone; or Ab-5(Clone DO-7) (amino acids 37 to 45; NEOMARKERS, INC., CA) which reactswith wild-type and mutant p53, according to the procedure of ReferenceExample 2. The same blot was reacted with anti-human mouse β-actinantibody (Sigma) to confirm total protein content.

[0133]FIG. 17A is the western blot showing the expression of p53 tumorsuppressor in HCCR-1H and parental wild-type 293 cells; and FIG. 17B isthe western blot showing β-actin of the same blot. As can be seen from17A, p53 protein levels were markedly elevated in HCCR-1H cells ascompared with the parental wild-type 293 cells.

[0134] Further, HCCR-1H cells obtained in Example 2 were cultured inmethionine-free RPMI 1640 medium containing 2% fetal calf serum and 2 mMglutamine (Sigma) for 2 hours and then labeled with 100 μl of L-³⁵Smethionine (Amersham). The cells were washed with 1×Hanks buffer (Gibco,USA), and then further cultured in normal RPMI 1640 medium containingL-methionine for 0.5, 1, 2 or 4 hours. The cells were dissolved in animmunoprecipitation solution (50 mM Tris[pH8.0], 5 mM EDTA, 150 mM NaCl,1% NP-40, 1 mM phenylmethylsulfonyl fluoride, 10 mM benzamidine, 1 μg ofpepstatin per ml, 10 mM sodium bisulfite), washed with 50 μl of proteinA-sepharose solution (Sigma) and then quantified. The cell lysate wassubjected to immunoprecipitation (Kessler, S. W., J. Immunol., 115,1617-1623 (1975)) using anti-p53 antibody followed by SDS-PAGE. Theabove procedure was repeated using Hep 3B hepatoma cells (ATCC HB-8064),as a control lacking p53 gene.

[0135]FIG. 18 is the immunoprecipitation result showing the expressionof p53 tumor suppressor in HCCR-1H and parental wild-type human 293cells. As can be seen from FIG. 18, p53 protein levels were markedlyelevated in HCCR-1H cells as compared with parental wild-type 293 cells.Therefore, it is believed that in the tumorigenesis mechanism induced byHCCR-1 protooncogene, p53 protein is highly expressed but remains in aninactive form which does not suppress the tumor.

[0136] (Step 2) Expression patterns of MDM2, bax, p21WAF1, p16IN4A andp14^(ARF) genes

[0137] Since p53 is a part of a regulatory loop that also involves MDM2,Bax and p14^(ARF), while directly influencing tumor suppressor genesp21WAF1 and p16INK4A, expression patterns of MDM2, Bax, p14^(ARF),p21WAF1 and p16INK4A were assessed.

[0138] Using HCCR-1H cells obtained in Example 2; and anti-MDM2,anti-Bax, anti-p21WAF1 and anti p16INK4A antibodies (Oncogene ResearchProducts, USA) and p14^(ARF) (Lab Vision, USA), western blot wasconducted according to the procedure of Reference Example 2. The sameblot was reacted with anti-β-actin antibody to confirm the total proteincontent.

[0139]FIGS. 19A, 19C, 19E, 19G and 19I are the western blot analysisresults showing the expression of MDM2, Bax, p21WAF1, p16INK4A andp14^(ARF), respectively, in HCCR-1H cell; and FIGS. 19B, 19D, 19F, 19Hand 19J are β-actin in each of the above blots. As can be seen fromFIGS. 19A, 19C, 19E and 19G, MDM2, Bax, p21WAF1, p16INK4A and p14^(ARF)protein levels are decreased compared with the parental wild-type 293cells. These results suggest that HCCR-1 overexpression may specificallyinhibit the transcription of MDM2, Bax, p21WAF1 and p16INK4A, therebyreducing the protein levels thereof.

[0140] In summarizing these results together with those of Step 1, it isbelieved that overexpression of HCCR-1 gene increases p53 stability butthe p53 protein does not form an autoregulatory loop which regulatesexpression of MDM2, bax and p21WAF1 positively. Particularly, as can beseen from FIGS. 19E and 19G, kinase inhibitor protein family p21WAF1 andp16INK4A were downregulated in HCCR-1H cells by a p53-independentmechanism. As can be seen from FIG. 19I, p14^(ARF) did not appear to beaberrantly expressed either in parental wild-type 293 nor in HCCR-1Hcells. These results suggest that alteration of the MDM2-p14^(ARF) loopis not a likely mechanism of p53 inactivation.

[0141] The above results together with those of Step 3 of Example 13indicate that the overexpression of HCCR-1 protooncogene both in mouseand human cells can affect cell cycle control and has a correlation withthe p53 and p21 cell cycle regulators.

[0142] (Step 3) Protein-protein interaction partner of HCCR-1

[0143] To determine a partner of HCCR-1 in the protein-proteininteraction, a yeast two-hybrid screen was conducted as follows.

[0144]E. coli JM-109/HCCR1 (KCTC 0667BP) cells were cultured andexpression vector pCEV-LAC/HCCR-1 was isolated therefrom. Expressionvector pCEV-LAC/HCCR-1 was cleaved with SalI and HCCR-1 protooncogenethus obtained was inserted at BamHI/SalI sites of yeast two-hybridvector (Clontech, USA) containing pLexA DNA-binding domain to obtainvector pLexA-HCCR-1 expressing a fusion protein of pLexA DNA-bindingdomain with HCCR-1 protein. Vector pLexA-HCCR-1 was mixed with yeastEGY48 (Clontech, USA) and heated at 42° C. for 10 min. to transform theyeast. The resulting yeast was transformed with human fetal brain cDNAlibrary fused with pB42AD containing pLexA-activating domain (Clontech,USA) in the same method. β-galactosidase filter lift assays wereperformed by replicaplating the transformant into Trp⁻, Leu⁻, His⁻selection plates. When a transformant has a partner gene derived fromcDNA library, the partner protein fused with pLexA-activating domainbinds to HCCR-1 protein fused with pLexA DNA-binding domain to form ablue colony on a plate medium containing X-gal. To eliminate falsepositives, yeast mating assay (Guarente, L., Proc. Natl. Acad. Sci. USA,90, 1639-1641 (1993)) was conducted.

[0145] From the clones thus obtained, a gene encoding a protein-proteininteraction partner of HCCR-1 was screened.

[0146] The colony thus obtained was cultured in glucose medium andvector containing the partner gene was extracted therefrom using glassbeads. The vector was introduced into E. coli KC8 (Clontech, USA) byelectroporation and the resulting E. coli KC8 cells were plated on M9minimal medium to select a transformant. Plasmid DNA was extracted fromthe transformant and E. coli DH5α was transformed with the plasmid DNAand cultured in LB medium to amplify the plasmid DNA. Plasmid DNA wasextracted from the culture and cleaved with HindIII to obtain 1 kbfragment containing the partner gene. The nucleotide sequence of thepartner gene was determined to identify TPD52L2 gene (GenBank accessionnumber NM003288). D52 protein encoded by TPD52L2 gene was originallyidentified through its elevated expression level in human breastcarcinoma, and play roles in calcium-mediated signal transduction andcell proliferation (Byrne, J. A., et al., Cancer Res., 55, 2896-2903(1995); and Byrne, J. A., et al., Oncogene, 16, 873-881 (1998)).

[0147] (Step 4) Co-transfection

[0148] In order to examine interaction of HCCR-1 and D52 proteins,co-transfection was conducted as follows.

[0149]E. coli JM-109/HCCR1 (KCTC 0667BP) cells were cultured andexpression vector pCEV-LAC/HCCR-1 was isolated therefrom. Expressionvector pCEV-LAC/HCCR-1 was cleaved with SalI and the full length HCCR-1cDNA thus obtained was inserted at SalI site of expression vectorN-terminal pFALG-CMV (Sigma, USA) to obtain expression vectorpFLAG-HCCR-1 expressing a fusion protein of FLAG with HCCR-1. TPD52L2gene obtained in Step 3 was inserted at NotI site of expression vectorpcDNA3 (Invitrogen, USA) to obtain expression vector pGST-TPD52L2expressing a fusion protein of GST with D52.

[0150] CHO cell was co-transfected with expression vectors pFLAG-HCCR-1and pGST-TPD52L2 and then cultured in RPMI 1640 medium. After 48 hours,the CHO cells were treated with trypsin and harvested. The CHO cellswere washed with PBS and then resuspended in IP buffer (10 mM Tris-HCl[pH 7.4], 150 mM NaCl, 1% NP40, 2 mM sodium vanadate, 10 mM sodiumfluoride, 10 mM sodium pyrophosphate, 5 mM EDTA, 10 μg/ml aprotinine, 10μg/ml leupeptine, 10 μg/ml pepstatine, 1 mM phemylmethylsulfonylfluoride). The cell suspension was then passed through a 27-gauge needleand the resulting cell lysate was spun to pellet the unbroken cells. Thesupernatant was precleared by mixing with preimmune IgG and protein Abeads (Sigma, USA). FLAG-HCCR-1 or GST-TPD52L2 fusion protein in thecell lysate was immunoprecipitated using anti-FLAG or anti-GST antibody(Sigma, USA) (Kesler, S. W., J. Immunol., 115, 1617-1623 (1975)). Theresidual protein samples were subjected to western blot using anti-GSTor anti-FLAG antibody according to the procedure of Reference Example 2.

[0151]FIGS. 20A and 20B are the western blot analysis results showingGST protein and PLAG protein, respectively. As can be seen from FIGS.20A and 20B, the fusion protein was detected in both precipitates. Theseresults suggest that D52 protein interacts with HCCR-1 protein.

EXAMPLE 15 Preparation of Transgenic Mouse

[0152] In order to ensure that the HCCR-1 protooncogene inducetumorigenesis in vivo, a transgenic mouse expressing HCCR-1protooncogene under the CMV promoter.

[0153] To obtain HCCR-1 cDNA useful in the preparation of a transgenicmouse, E. coli JM-109/HCCR1 cells (KCTC 0667BP) were cultured andexpression vector pCEV-LAC/HCCR-1 was isolated therefrom. Expressionvector pCEV-LAC/HCCR-1 was cleaved with SalI and the full length HCCR-1cDNA thus obtained was inserted at XhoI site of expression vector pcDNA3to obtain expression vector pcDNA3-HCCR-1. Expression vectorpDNA3-HCCR-1 was cleaved with SalI and XmnI, electrophoresed on 0.7%agarose gel, and isolated by electroelution. The isolated DNA fragmentwas dialyzed against 10 mM Tris-HCl (pH 7.4)/0.2 mM EDTA solution andadjusted to a final concentration of 4 ng/ml. The structure of theresulting DNA contains CMV promoter, HCCR-1 cDNA gene and bovine growthhormone (bGH) polyA sequence as shown in FIG. 21.

[0154] The DNA was microinjected into a zygote of mouse FVB/N strain(Samyuk Corp., CAMTAKO, Korea) according to pronuclear DNAmicroinjection method (Gordon, J. W., et al., Pro. Natl. Acad. Sci. USA,77, 7382-7384 (1980)): The DNA solution was microinjected into thepronucleus of 1-cell stage zygote and the resulting zygote was incubatedfor 20 hours to select the 2-cell stage embryo. The 2-cell stage embryowas implanted into oviduct of pseudopregnant, recipient ICR mouse(Samyuk Corp., CAMTAKO, Korea). The progeny mice were obtained from therecipient mouse, and DNA samples obtained by tail biopsy of the progenymice were subjected to southern blot analysis.

[0155]FIG. 22 is the transgenic mouse derived from the embryo introducedwith HCCR-1 protooncogene. As can be seen from FIG. 22, the tumor mass,measuring about 1.5 cm×1.5 cm, is noted in the right axillary areaadjacent to the breast.

[0156] The tumor was isolated and observed with the naked eye.

[0157]FIG. 23 is the photograph of the breast tumor of transgenic mouse.As can be seen from FIG. 23, the tumor of transgenic mouse is single,well-circumscribed nodule without capsule.

[0158] To examine the morphological features of the tumor, the tumor wasstained with hematoxylin-eosin. FIG. 24 is the hematoxylin-eosinstaining result of the tumor. As can be seen from FIG. 24, the tumorconsists of papillary structures and glandular or duck-like structures.Extensive necrosis is noted in the central portion. Normal breast tissueis evident adjacent to the tumor. Cuboid or ovoid cells have indistinctborder and a moderate amount of granular, eosinophilic cytoplasm. Thetumor cells have an large, pleomorphic and hyperchromatic nuclei andmany mitoses, including some atypical forms. Small but distinct nucleoliare noted. These results suggest that the tumor of transgenic mouseexhibits characteristics of ductal papillary adenocarcinoma of breast.

[0159]FIG. 25 is the transmission electron microscope picture of thetumor, wherein the scale bar represents the length of 2 μm. As can beseen from FIG. 25, ovoid tumor cells have abundant cytoplasmicorganelles. Nuclei have clumping and margination of chromatin anddistinct nucleoli. Cytoplasm is filled with some mitochondria and manyvesiculated rER. Some desmosomes are evident.

[0160] The embryo of the transgenic mouse was designated HCCR-1, whichwas deposited on Dec. 26, 2000 with the Korean Collection for TypeCultures under the accession number of KCTC 0924BP.

EXAMPLE 16 Expression of HCCR-1 Protooncogene in Breast, Kidney, Ovaryand Stomach Tumor Tissues

[0161] To examine the expression of HCCR-1 protooncogene in breast,kidney, ovary and stomach tumor tissues, total RNA was isolated from thebreast, kidney, ovary and stomach tumor tissues according to theprocedure of Reference Example 4 and subjected to northern blot analysisaccording to the procedure of Reference Example 5 using ³²P-labeledrandom HCCR-1 cDNA probe. For the comparison, the above procedures wererepeated using normal breast, kidney, ovary and stomach tissues.

[0162]FIG. 26A is the northern blot analysis result showing theexpression of HCCR-1 protooncogene in breast, kidney, ovary and stomachtumor tissues; and FIG. 26B is the same blot hybridized with β-actinprobe. As can be seen from FIGS. 26A and 26D, the human breast, kidney,ovary and stomach tumor tissues showed increased expression of HCCR-1protooncogene when compared with the normal tissues.

[0163] To examine the HCCR-1 protein expressed in the breast, kidney,ovary and stomach tumor tissues, the breast, kidney, ovary and stomachtumor tissues were subjected to western blot analysis using anti-HCCR-1serum obtained in Preparation Example 2 according to the procedure ofReference Example 2.

[0164]FIG. 27 is the western blot analysis result showing the expressionof HCCR-1 protooncogene in human breast, kidney, ovary and stomachcancer tissues. As can be seen from FIG. 27, the human breast, kidney,ovary and stomach tumor tissues showed increased expression of 50 kDaHCCR-1 protein expression when compared with their normal counterparts,respectively.

EXAMPLE 17 Chromosomal Localization of HCCR-1 Protooncogene

[0165] The full length HCCR-1 cDNA obtained in Preparation Example 1 waslabeled using random prime labeling kit (Promega, USA) to obtain aprobe. Human placenta Lambda Genomic Library (Stratagene, USA) wasscreened using the probe to obtain 20 kb genomic DNA.

[0166] The genomic DNA was subjected to fluorescence in situhybridization (FISH) (Cherif, D., et al., Proc. Natl. Acad. Sci. USA,87, 6639-6649 (1990)): The genomic DNA on a slide was labeled withSpectrumRed-dUTP using nick translation kit (Vysis, USA). The slide wasobserved under a Zeiss fluorescence microscope. Chromosomes werecounterstained with DAPI (Sigma, USA).

[0167]FIG. 28 is the fluorescence in situ hybridization analysis resultshowing that HCCR-1 protooncogene is located on long arm (12q) of 12thchromosome.

[0168] While the subject invention has been described and illustratedwith reference to the preferred embodiments only, it may be apparent tothose skilled in the art that various changes and modifications can bemade therein without departing from the spirit and scope of the presentinvention which is defined in the appended claims.

1 5 1 2118 DNA Homo sapiens CDS (9)..(1088) sig_peptide (9)..(83)misc_feature (435)..(494) transmembrane domain 1 ctgtgaag atg gcg ctctcc agg gtg tgc tgg gct cgg tcg gct gtg tgg 50 Met Ala Leu Ser Arg ValCys Trp Ala Arg Ser Ala Val Trp 1 5 10 ggc tcg gca gtc acc cct gga catttt gtc acc cgg agg ctg caa ctt 98 Gly Ser Ala Val Thr Pro Gly His PheVal Thr Arg Arg Leu Gln Leu 15 20 25 30 ggt cgc tct ggc ctg gct tgg ggggcc cct cgg tct tca aag ctt cac 146 Gly Arg Ser Gly Leu Ala Trp Gly AlaPro Arg Ser Ser Lys Leu His 35 40 45 ctt tct cca aag gca gat gtg aag aacttg atg tct tat gtg gta acc 194 Leu Ser Pro Lys Ala Asp Val Lys Asn LeuMet Ser Tyr Val Val Thr 50 55 60 aag aca aaa gcg att aat ggg aaa tac catcgt ttc ttg ggt cgt cat 242 Lys Thr Lys Ala Ile Asn Gly Lys Tyr His ArgPhe Leu Gly Arg His 65 70 75 ttc ccc cgc ttc tat atc ctg tac aca atc ttcatg aaa gga ttg cag 290 Phe Pro Arg Phe Tyr Ile Leu Tyr Thr Ile Phe MetLys Gly Leu Gln 80 85 90 atg tta tgg gct gat gcc aaa aag gct aga aga ataaag aca aat atg 338 Met Leu Trp Ala Asp Ala Lys Lys Ala Arg Arg Ile LysThr Asn Met 95 100 105 110 tgg aag cac aat ata aag ttt cat caa ctt ccatac cgg gag atg gag 386 Trp Lys His Asn Ile Lys Phe His Gln Leu Pro TyrArg Glu Met Glu 115 120 125 cat ttg aga cag ttc cgc caa gac gtc acc aagtgt ctt ttc cta ggt 434 His Leu Arg Gln Phe Arg Gln Asp Val Thr Lys CysLeu Phe Leu Gly 130 135 140 att att tcc att cca cct ttt gcc aac tac ctggtc ttc ttg cta atg 482 Ile Ile Ser Ile Pro Pro Phe Ala Asn Tyr Leu ValPhe Leu Leu Met 145 150 155 tac ctg ttt ccc agg caa cta ctg atc agg catttc tgg acc cca aaa 530 Tyr Leu Phe Pro Arg Gln Leu Leu Ile Arg His PheTrp Thr Pro Lys 160 165 170 caa caa act gat ttc tta gat atc tat cat gctttc cgg aag cag tcc 578 Gln Gln Thr Asp Phe Leu Asp Ile Tyr His Ala PheArg Lys Gln Ser 175 180 185 190 cac cca gaa att att agt tat tta gaa aaggtc atc cct ctc att tct 626 His Pro Glu Ile Ile Ser Tyr Leu Glu Lys ValIle Pro Leu Ile Ser 195 200 205 gat gca gga ctc cgg tgg cgt ctg aca gatctg tgc acc aag ata cag 674 Asp Ala Gly Leu Arg Trp Arg Leu Thr Asp LeuCys Thr Lys Ile Gln 210 215 220 cgt ggt acc cac cca gca ata cat gat atcttg gct ctg aga gag tgt 722 Arg Gly Thr His Pro Ala Ile His Asp Ile LeuAla Leu Arg Glu Cys 225 230 235 ttc tct aac cat cct ctg ggc atg aac caactc cag gct ttg cac gtg 770 Phe Ser Asn His Pro Leu Gly Met Asn Gln LeuGln Ala Leu His Val 240 245 250 aaa gcc ttg agc cgg gcc atg ctt ctc acatct tac ctg cct cct ccc 818 Lys Ala Leu Ser Arg Ala Met Leu Leu Thr SerTyr Leu Pro Pro Pro 255 260 265 270 ttg ttg aga cat cgt ttg aag act cataca act gtg att cac caa ctg 866 Leu Leu Arg His Arg Leu Lys Thr His ThrThr Val Ile His Gln Leu 275 280 285 gac aag gct ttg gca aag ctg ggg attggc cag ctg act gct cag gaa 914 Asp Lys Ala Leu Ala Lys Leu Gly Ile GlyGln Leu Thr Ala Gln Glu 290 295 300 gta aaa tcg gct tgt tat ctc cgt ggcctg aat tct acg cat att ggt 962 Val Lys Ser Ala Cys Tyr Leu Arg Gly LeuAsn Ser Thr His Ile Gly 305 310 315 gaa gat agg tgt cga act tgg ctg ggagaa tgg ctg cag att tcc tgc 1010 Glu Asp Arg Cys Arg Thr Trp Leu Gly GluTrp Leu Gln Ile Ser Cys 320 325 330 agc ctg aaa gaa gct gag ctg tct ctcttg ctg cac aac gtg gtc ctg 1058 Ser Leu Lys Glu Ala Glu Leu Ser Leu LeuLeu His Asn Val Val Leu 335 340 345 350 ctc tcc acc aac tac ctt ggg acaagg cgc tg aatgaaccat ggagcggat 1110 Leu Ser Thr Asn Tyr Leu Gly Thr ArgArg 355 360 gcattgtcct gcagtcgtat agtatagcag tgcaggaaca aacagcacttgccagcaaag 1170 tctgtgtgta ctgttaagtg tgtgggaggc agagagagga gcaggggccatgggcttcac 1230 agcatggcac acctgtggga actgcagaca ttcctctcac agctagaactgaaacaaacc 1290 ctcttgctag gggtggtccg tgtgaggtgt catcctgtcc ccctcataattactaatagc 1350 tggaactggc agcagcctct actgggcttt tactgtgatg tgttcagttcatgtcctagg 1410 aagtcagctt ttgccccagg tgggaatcct tatttggctt aggactgatccacttccatg 1470 ttacttacat ctgtgggttt ttgttgttgc tgttagaaaa tttttggctggtgaaaacag 1530 cactcctttg gctggagcac ttgtgtccat gcatgtactt gggtgtttccctccatcctt 1590 tctgatatga ccaaaaatca agttgttttg ttttttgtca ccttcactggcatgggctaa 1650 ccacttcttt ttcaaaccct ctgaacacct ttttctgatg ggtaacttgcaggaatattc 1710 tattggaaaa gataacagga agtacaagtg cttcttgacc ccttcctcaatgtttctagc 1770 cttcactctc cattgtcttt tctgggctgt attacagccc tctgtggatcttcaactctg 1830 ctgcctccac tgtgatgcag cagtccaact gtaactgaca gtggctgccttctctgggcc 1890 atggatcaca cctgtaaggt actaattact gcccagcctg gggagatcaggagaggtctg 1950 catagttagt aagttgggtt tagcttttgt gtgtgcatca gtgacttagagttctgtaat 2010 aacttattgt aaatgcatga agcactgttt ttaaacccaa gtaaagactgcttgaaacct 2070 gttgatggaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaa2118 2 360 PRT Homo sapiens 2 Met Ala Leu Ser Arg Val Cys Trp Ala ArgSer Ala Val Trp Gly Ser 1 5 10 15 Ala Val Thr Pro Gly His Phe Val ThrArg Arg Leu Gln Leu Gly Arg 20 25 30 Ser Gly Leu Ala Trp Gly Ala Pro ArgSer Ser Lys Leu His Leu Ser 35 40 45 Pro Lys Ala Asp Val Lys Asn Leu MetSer Tyr Val Val Thr Lys Thr 50 55 60 Lys Ala Ile Asn Gly Lys Tyr His ArgPhe Leu Gly Arg His Phe Pro 65 70 75 80 Arg Phe Tyr Ile Leu Tyr Thr IlePhe Met Lys Gly Leu Gln Met Leu 85 90 95 Trp Ala Asp Ala Lys Lys Ala ArgArg Ile Lys Thr Asn Met Trp Lys 100 105 110 His Asn Ile Lys Phe His GlnLeu Pro Tyr Arg Glu Met Glu His Leu 115 120 125 Arg Gln Phe Arg Gln AspVal Thr Lys Cys Leu Phe Leu Gly Ile Ile 130 135 140 Ser Ile Pro Pro PheAla Asn Tyr Leu Val Phe Leu Leu Met Tyr Leu 145 150 155 160 Phe Pro ArgGln Leu Leu Ile Arg His Phe Trp Thr Pro Lys Gln Gln 165 170 175 Thr AspPhe Leu Asp Ile Tyr His Ala Phe Arg Lys Gln Ser His Pro 180 185 190 GluIle Ile Ser Tyr Leu Glu Lys Val Ile Pro Leu Ile Ser Asp Ala 195 200 205Gly Leu Arg Trp Arg Leu Thr Asp Leu Cys Thr Lys Ile Gln Arg Gly 210 215220 Thr His Pro Ala Ile His Asp Ile Leu Ala Leu Arg Glu Cys Phe Ser 225230 235 240 Asn His Pro Leu Gly Met Asn Gln Leu Gln Ala Leu His Val LysAla 245 250 255 Leu Ser Arg Ala Met Leu Leu Thr Ser Tyr Leu Pro Pro ProLeu Leu 260 265 270 Arg His Arg Leu Lys Thr His Thr Thr Val Ile His GlnLeu Asp Lys 275 280 285 Ala Leu Ala Lys Leu Gly Ile Gly Gln Leu Thr AlaGln Glu Val Lys 290 295 300 Ser Ala Cys Tyr Leu Arg Gly Leu Asn Ser ThrHis Ile Gly Glu Asp 305 310 315 320 Arg Cys Arg Thr Trp Leu Gly Glu TrpLeu Gln Ile Ser Cys Ser Leu 325 330 335 Lys Glu Ala Glu Leu Ser Leu LeuLeu His Asn Val Val Leu Leu Ser 340 345 350 Thr Asn Tyr Leu Gly Thr ArgArg 355 360 3 18 DNA Artificial Sequence oligonucleotide whichspecifically binds to HCCR-1 mRNA 3 cctggacatt ttgtcacc 18 4 25 DNAArtificial Sequence PCR primer 4 aggcaactag gatccaggca tttct 25 5 29 DNAArtificial Sequence PCR primer 5 gtcgacgcag ttcccacagg tgtgccatg 29

What is claimed is:
 1. A mammalian cell transformed with an expressionvector comprising a human protooncogene which has the nucleotidesequence of SEQ ID NO:
 1. 2. The mammalian cell of claim 1, which ismouse cell line HCCR-1M (KCTC 0923BP) or human cell line HCCR-1H (KCTC0922BP).
 3. A mammalian embryo carrying an introduced nucleic acidconstruct comprising a human protooncogene which has the nucleotidesequence of SEQ ID NO:
 1. 4. The mammalian embryo of claim 3, whereinthe mammal is mouse FVB.
 5. The mammalian embryo of claim 4, which ismouse embryo HCCR-1 (KCTC 0924BP)
 6. A transgenic cancered mammalderived from the embryo of any one of claims 3 to
 5. 7. The transgenicmammal of claim 6, wherein the cancer is ductal papillary adenocarcinomaof breast.
 8. A kit for diagnosing a cancer selected from the groupconsisting of breast, kidney, ovary and stomach cancers, which comprisesa probe having the nucleotide sequence complementary to mRNA transcribedfrom the human protooncogene having the nucleotide sequence of SEQ IDNO: 1 or a portion of the mRNA; or an antibody binding specifically to aprotein translated from the mRNA or a portion of the protein.